The assignment of the proton and carbon spectra of the cyclic peptide cyclo(-Phe1-Pro2-Thr3-Lys(Z)'-TrpS- was accomplished by the application of multiple quantum proton-detected heteronuclear correlation spectroscopy. Since the proton spectrum shows severe overlap, the carbon chemical shifts were used to disentangle the proton resonances. The methodology described is useful even in cases where only limited quantities of materials are available.The combination of (i) a proton decoupled C,H correlation spectrum and (ii) a (not decoupled) H-relayed C,H correlation gave the assignments of all CH, groups. The non-protonated carbons, i.e. the carbonyl carbons of the peptide bond, were assigned with a C,H correlation optimized for long-range couplings, an experiment that also gave helpful information about the conformational features of the hexapeptide.The cyclic peptide contains a Phe-Pro cis-peptide bond forming a @VI-like bend and a 8-turn about the amino acids Thr-Lys-TrpPhe. Although the conventional discussion of NMR parameters indicates a strong preference for one conformation, the quantitative evaluation of NOEderived distances in restrained MD calculations proves that the type of 8-turn in the last-mentioned region is not unique. Whereas the MD calculations converge to a 811' turn, the vicinal proton coupling constants are in better agreement with type 81. Thus a dynamic equilibrium of the backbone is proposed.KEY WORDS Cyclic peptide Inverse spectrum Long-range proton-carbon couplings Inverse relayed spectrum NMR assignments Peptide conformation
The solution conformation of the 27-residue polypeptide hormone secretin in dimethyl sulfoxide has been determined on the basis of 'H-NMR measurements. The experimental data set used in the structure determination consisted of 98 nuclear-Overhauser-enhancement-derived interproton and dihedral angle restraints from coupling constants. The NH-NH and Ha-NH NOES were determined from build-up rates, while the remaining distances were classified in a qualitative manner.The structure calculations consisted of two phases. First, dynamical simulated annealing calculations were carried out to find conformations of the peptide which satisfy NOE and $ J dihedral restraints. The convergence of ten calculated structures was good except for those regions of the molecule where NOE data were not unambiguous. From the calculated set another initial structure was built which was again minimized in several 5-ps calculations now employing the full empirical energy function. The resulting structures of secretin reveal conformationally well-defined regions, but not a single uniform secondary structure. The structure is different from the calculated structure from trifluoroethanol/water measurements.Secretin is a gut hormone of the glucagon family [l] and exhibits various functions as major regulator of pancreatic exocrine secretion 121. Beside these actions it shows inhibitorial effects of gastric acid secretion and gastrin release, and it influences the release of insulin and growth hormone releasing factor [3].It is known that under conditions of reduced water activity many hormones prefer an ordered structure. So the residue 1 -29 fragment of growth hormone releasing factor and analogues of the hormone adopt a complete a-helical conformation in 30 -40% trifluoroethanol solution 14, 51. A similar secondary structure is preferred for glucagon and secretin [6 -81. Therefore it is reasonable to expect that sequentially related hormones of the glucagon family may also exhibit helical conformation under these conditions. If one takes into account the broad spectrum of biological activities of these natural compounds, it seems important to study their conformations under experimental conditions that emphasize variations in the conformations rather than similarities. The solution structure of growth hormone releasing factor in dimethyl sulfoxide (Me,SO) differs strongly from the conformation in trifluoroethanol/water, where it exhibits a completely helical backbone 1. 51. Since trifluoroethanol/water is known to favour such a helical secondary structure, the purpose of the following work was to analyse the conformation of secretin in Me2S0, and to point out differences between the conformations in trifluoroethanol/water and Me2S0.In this paper we present the Me2S0 solution structure of secretin using 'H-NMR data as input for dynamical simulated annealing and restrained molecular dynamics calculations. The assignments of the 'H resonances was achieved by means of correlated [9,, such as correlated spectroscopy (COSY) and relayed correlated...
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